Scientists may have been able to create Dolly the sheep, but researchers at Pitt have… Scientists may have been able to create Dolly the sheep, but researchers at Pitt have found it nearly impossible to create a clone of Dolly Parton.

Researchers at the Pittsburgh Development Center at the Magee-Womens Research Institute have found that fundamental flaws in embryonic development may make therapeutic cloning of non-human primates difficult and the reproduction of both human and non-human primates impossible.

Their study, which was funded by the National Institute of Child Health, the National Center for Research Resources, the National Institute of Environmental Health sciences and private philanthropies, was published last week in the journal Science.

In therapeutic cloning, scientists stimulate limited cell division in an unfertilized egg cell to produce embryonic stem cells. These cells can later differentiate into different types of body tissue.

Reproductive cloning involves scientists injecting a donor nucleus into an egg cell and then implanting that egg into a living surrogate female in an attempt to create pregnancy.

Gerald Schatten, director of the PDC and vice chairman and professor in Pitt’s obstetrics, gynecology, reproductive sciences and cell biology departments, used nuclear transfer methods on 724 eggs retrieved from female rhesus monkeys. Nuclear transfer involves taking the nucleus from a somatic, or body, cell and inserting it into an egg without a nucleus. The nuclear transferring process causes the new cell to exhibit the properties of the donor nucleus.

Though 33 of the embryos produced through nuclear transfer were implanted into female monkeys after initial cell division, no pregnancies occurred. Scientists, when examining images of DNA and cell structure, found that while cell division occurred in a superficially normal manner, each individual cell had chromosomal problems.

“We used antibodies to tag the cell proteins and DNA so that we could track progress,” Calvin Simerly, associate professor of obstetrics, gynecology, and reproductive sciences at Pitt and the paper’s first author “When cells divide, there are very basic things that are supposed to happen, and they just didn’t happen.”

Christopher Navara, another researcher, explained that many of these cloning difficulties are because of missing motor proteins, which form the mitotic spindle, in the cells. The mitotic spindle is the structure in a cell responsible for organizing DNA prior to cell division and then separating chromosomes and the resulting daughter cells during division. This separation is necessary for the cell to be able to replicate itself successfully.

Navara added that during the cloning of non-human primates, scientists remove the DNA and consequently take about 5 to 10 percent of the egg in the process. In non-human primates, many of those motor proteins are collected around the DNA, so they are often removed along with that DNA.

When examining cells that were formed using nuclear transfer techniques, researchers found that spindles had unequal chromosome counts and chaotic structures. Without a sufficient amount of motor proteins, the mitotic spindle does not form properly, which causes some of the daughter cells to have abnormal number of chromosomes.

Navara explained that the reason cloning has been successful in non-primate animals is because motor proteins are evenly distributed throughout the egg, as evidenced in eggs taken from cows.

The research team’s current goal is working toward finding a way for modified eggs of non-human primates to get through the first few cell divisions so they can ultimately be used to make identical animal models or embryonic stem cells for study. Navara added that even if only 10 percent of cells in the new embryo were normal, scientists may be able to use them.

“It would be easier if there weren’t these problems, but we may still be able to isolate the embryonic stem cells,” he said.

Though the PDC supports an international ban on human reproductive cloning, it is pursuing new therapeutic cloning techniques to produce human embryonic stem cells to treat Parkinson’s disease, diabetes and strokes. By further developing nuclear transfer technology in non-human primates the researchers hope to create relevant identical animal models to study human diseases or possible uses of embryonic stem cells. The PDC is also responsible for creating the first non-human primate through embryo splitting and the first genetically modified non-human primate.

Schatten, the principal researcher of the study, maintains a positive outlook on the Center’s cloning research.

“Current techniques such as those used to create Dolly the sheep, mice and other domestic animals do not work in non-human primates,” he said. “I don’t want to say that this will never work. Given enough time and materials, we may discover how to make it work. It just doesn’t work now.”